1. Field of the Invention
The field of the invention is markers for identifying transgenic animals.
2. Background of the Invention
Genetic manipulation of insects and other arthropods is a highly desirable goal for the development of better control strategies to fight agricultural pests and disease vector species. Transposon-based transformation techniques had been available for Drosophila, but only recently did the discovery of new transposons enable this approach in other insects (O""Brochta and Atkinson, 1996, Insect Biochem. Molec. Biol. 26, 739-53), i.e medflies (Loukeris et al., 1995, Science 270, 2002-5; Handler et al., 1998, PNAS 95, 7520-5) and mosquitoes (Coates et al., 1998, PNAS 95, 3748-51; Jasinskiene et al., 1998, PNAS 95, 3743-7). However, a major obstacle in the use of these transposons has been the difficulty to obtain marker genes that allow easy and reliable identification of transgenic animals. In fact, a main reason why germline transformation experiments have not been carried out routinely so far in non-dipteran insects, is the lack of specific markers to follow gene transfer (DeVault et al., 1996, Genome Research 6, 571-9). Here we present a novel marker system broadly suitable for eye-bearing animals.
In combination with a set of promiscuous vectors, our system permits the study of biologically relevant questions in almost any species, not only in established model organisms. Since the very same system can be used in a series of different organisms, comparative biological and functional evolutionary studies are facilitated, providing a vital tool for the emerging field of evolutionary developmental biology. Furthermore, expression in the eyes allows visualization of the signal in animals with non-transparent cuticle, and transgenic animals can be identified as larvae, pupae and adults. Together with the fact that the system can be applied to competitive wild type strains rather than potentially labile mutant lines, makes the system particularly applicable to pest management programs.
The subject methods generally comprise (a) introducing into a genome of an animal a genetic construct comprising a transcriptional regulatory element operably linked to a heterologous marker gene encoding a marker, wherein the element drives expression of the marker across genera transgenic in the construct sufficient to visually detect the marker in photoreceptive cells or organs, and (b) selecting for transgenesis by visually detecting the marker in a photoreceptive cell or organ of the animal. In particular embodiments, the construct comprises a vector, such as transposon or retrovirus, particularly a polytropic vector. The construct may integrate into the genome by homologous or non-homologous recombination. In particular embodiments, the transcriptional regulatory element comprises a binding site selected from a Pax-6 binding site, a Glass binding site, etc., particularly a plurality of P3 sites, and the marker is a fluorescent protein, particularly a green fluorescent protein or variant thereof.
The subject compositions include polytropic vectors functional in nondipteran species and comprising a transcriptional regulatory element operably linked to a heterologous marker gene encoding a marker, wherein the element drives expression of the marker across genera transgenic in the construct sufficient to visually detect the marker in photoreceptive cells or organs, particularly wherein the marker is the only visually detectable indicator of transgenesis encoded by the vector. The invention also provides cells and animals transgenic in the subject constructs and/or made by the subject methods.
The following descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms xe2x80x9caxe2x80x9d and xe2x80x9canxe2x80x9d mean one or more, the term xe2x80x9corxe2x80x9d means and/or and polynucleotide sequences are understood to encompass opposite strands as well as alternative backbones. The subject methods and applications are applicable to a wide variety of photoreceptor cell or organ bearing animals. By photoreceptive cells or organs is meant any light sensing cell or organ of an animal and include cells such as simple pigmented light sensitive cells or retinular cells and structures such as ocelli also called simple eyes or eye spots like the direct or inverted pigment cups of many worms, structures such as compound eyes found in many arthropods, structures such as complex eyes or camera eyes of cephalopod molluscs and vertebrates. Unless otherwise noted, the term eye is used herein to collectively refer to these various light sensing cells or organs. The suitability of any particular photoreceptor cell or organ bearing animal is readily determined empirically, using conventional genetic transformation procedures and screening procedures, as exemplified below. Genera demonstrating transgenesis according to the disclosed methods include vertebrates, particularly mammals, fish and birds, and non-arthropod and arthropod invertebrates, such as Crustacea, Chelicerata and Insecta, such as Diptera such as flies and mosquitoes, and non-dipteran insects, such as Lepidoptera, Hymenoptera, Coleoptera, Neuroptera, Hemiptera, Isoptera, Dictyoptera, and Orthoptera.
The subject methods employ a transcriptional regulatory element operably linked to a heterologous marker gene encoding a marker, wherein the element drives expression of the marker across genera transgenic in the construct sufficient to visually detect the marker in photoreceptive cells or organs. By drives expression across genera is meant that the element is capable of promoting gene expression in a plurality of genera, preferably including a non-dipteran insect, more preferably including a non-insect arthropod. Preferred elements are functional in a plurality of taxonomic (Zoological Record, BIOS UK, 1999) families, preferably a plurality of orders, more preferably a plurality of classes, more preferably a plurality of phyla. In particular embodiments, the element is functional in at least the families Drosophilidae, Calliphoridae and Culicidae, preferably in the orders Diptera, Lepidoptera and Coleoptera, more preferably in the classes Insecta, Malocostraca and Chelicerata, even more preferably across the phyla Arthropoda, Mollusca and Chordata.
To drive marker expression in a series of diverged organisms requires a promoter which is active in a wide range of species. Furthermore, to avoid problems with low expression and the interference of autofluorescence, a regional specific promoter is preferable over a constitutively active one. A wide variety of regulatory elements may be employed, so long as they meet the requisite functional limitations. These may be natural promoter elements, naturally driving gene expression in photoreceptive cells or organs, elements derived from such natural promoter elements by mutational selection or consensus sequences, synthetic elements derived by iterative selection process, e.g. SELEX procedures, etc. In a particular embodiment, the element comprises a binding site selected from a Pax-6, a Pax-6 like binding site such as a twin-of-eyeless (TOY) binding site, a Glass binding site, etc. In more particular embodiments, the element comprises a Pax-6 Paired Domain or Homeodomain binding site, more particularly a P3 site, wherein the P3 site comprises the sequence: TAATYNRATTA (SEQ ID NO:01), wherein Y=C or T; R=G or A; N=any nucleotide (Wilson et al., 1993, Genes Dev 7, 2120-34; Czerny and Busslinger, 1995, Mol Cell Biol 15, 2858-71). Tables 1-6 provide other exemplary transcriptional regulatory element binding sites functional in the subject methods. Pax-6 binding sites are of particular interest due to the evolutionary conserved role Pax-6-homologs play in eye development across different phyla (Callaerts et al., 1997, Annu Rev Neurosci 20, 483-532).
The strength and/or specificity of the element may often be enhanced by multimerizing the binding site, i.e. providing a plurality of binding sites within the element. The number of binding sites is readily optimized empirically and is generally from 3 to 9. The plurality may be directly linked or separated by spacer sequence of 1 bp to 1 kb, preferably fewer than 250 bp, more preferably fewer than 50 bp, which may be of any sequence compatible with the required functionality of the element. Exemplary spacers used herein include GAGAC, GAGC, and GGATCCAAGCTTATCGATTTCGAACCCTCGACCGCCGGAG (SEQ ID NO:44).
The element generally also comprises a basal RNA Pol II promoter, the core promoter site that generally contains a TATA box sequence and transcriptional initiation site, and which functions in conjuction with transcription enhancer functions provided in the subject elements by the transcription factor binding site regions. A wide variety of basal promoters may be employed in the elements so long as they facilitate, in conjunction with the binding site(s), the requisite transcriptional regulation. Exemplary basal promoter elements include those of the Drosophila hsp70 gene promoter and Adenovirus major late promoter (MLP).
A wide variety of multimeric and/or combinatorial binding sitexe2x80x94spacerxe2x80x94basal promoter combinations may be usedxe2x80x94essentially any combination functions in conjunction to provide the requisite transcriptional regulation. For best results we find 0 to 10 bp spacers between the binding sites provide optimal synergistic effect of the binding sites and a spacer of 20 to 40 bp should separate the binding sites from the basal promoter.
The construct includes a marker gene encoding a marker which, when expressed in the transgenic animal, is visually detectable in a photoreceptive cell or organ of the animal. Criteria for marker selection include detectability, physiological and method compatibility, e.g. smaller sized marker genes enable small transposon constructs resulting in high transformation rates. A wide variety of markers may be encoded, including ribozymes or protein enzymes such as galactosidase, luciferase (e.g. Wilson and Hastings, 1998, Annu Rev Cell Dev Biol 14, 197-230), etc., and particularly directly detectable proteins, more particularly fluorescent proteins, especially commercially available enhanced fluorescent proteins (e.g. EGFP, ECFP and EYFP, Clontech Laboratories, Inc.).
Fluorescent proteins may comprise naturally occurring, engineered (i.e., analogs) and/or synthetic sequences. For example, many cnidarians use natural green fluorescent proteins (xe2x80x9cGFPsxe2x80x9d) as energy-transfer acceptors in bioluminescence. Natural GFPs have been isolated from numerous animals, including the Pacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renilla reniformis, and Phialidium gregarium; Ward et al., Photochem. Photobiol., 35:803-808 (1982); Levine et al., Comp. Biochem. Physiol., 72B:77-85 (1982). In addition, a variety of Aequorea-related fluorescent proteins having useful excitation and emission spectra have been engineered by modifying the amino acid sequence of a naturally occurring GFP from Aequorea victoria (Prasher et al., Gene, 111:229-233 (1992); Heim et al., Proc. Natl. Acad. Sci., USA, 91:12501-04 (1994). Particularly useful are GFPs from or which derive from the jellyfish A. victoria (see e.g. U.S. Pat. No. 5,491,084 for applicable such GFPs) and include variants offering a variety of different excitation and emission wavelengths; see e.g. Heim and Tsein, 1996, Current Biology 6, 178-182. Exemplary amino acid variants include F64L, S65T, Y66W, N146I, M153T, V163A and N212K, and combinations thereof. For example, CFP is the GFP of Aequorea victoria with the following additional mutations: F64L, S65T, Y66W, N146I, M153T, V163A, N212K (Miyawaki et al., 1997, Nature 388:882-7), and YFP is the GFP of A. victoria with the following additional mutations: S65G, V68L, S72A, T203Y (Cubitt et al., 1999, Methods Cell Biol 58, 19-30). Accordingly, in preferred embodiments, the marker is a Aequorea or Aequorea-related fluorescent protein, see U.S. Pat. No. 5,912,137 for applicable sequence, scope, definitions and examples.
Suitable fluorescent proteins may also derive from other sources, and include the yellow fluorescent protein from Vibrio fischeri strain Y-1 (Baldwin et al., Biochemistry (1990) 29:5509-15) which requires flavins as fluorescent co-factors; Peridinin-chlorophyll, a red fluorescing binding protein from the dinoflagellate Symbiodinium sp. (Morris et al., Plant Mol Biol, (1994) 24:673:77); phycobiliproteins from marine cyanobacteria such as Synechococcus, e.g., phycoerythrin and phycocyanin (Wilbanks et al., J. Biol. Chem. (1993) 268:1226-35), yellow to red fluorescing proteins which require phycobilins as fluorescent co-factors.
The subject constructs may be introduced into the genome of the targeted animal by any convenient methods. A wide variety of transformation methods are widely known for and/or are adaptable to a wide variety of target animals. For example, many applications are amenable to direct injection of naked construct DNA. In other embodiments, the construct further comprises regions which provide homologous recombination and integration into a target site of the genome. In yet other embodiments, the constructs are incorporated in a vector, such as a transposon or retrovirus. A wide variety of vectors may be employed, as influenced by the nature of the construct and the targeted host. In more particular embodiments, the vector is polytropic (functional across multiple genuses) or pan tropic (functional across mutliple families, preferably classes, more preferably orders), a wide variety of which are well known in the art, including the following: (a) recombinant retroviruses for example Moloney murine leukemia virus-based vectors (here termed xe2x80x9cMLV greater than VSVGxe2x80x9d) having the envelope protein of vesicular stomatitis virus substituted for the amphotropic envelope protein (Burns et al., In Vitro Cell Dev Biol Anim (1996) 32:78-84; Lin et al., Science (1994) 265:666-669; Lu et al., (1996) PNAS 93:3482-3486; Jordan et al., (1998) Insect Mol Biol 7:215-222; U.S. Pat. No. 5,670,354; WO9603034); (b) recombinant baculoviruses for example vectors based on Autographa Californica Nuclear Polyhedrosis Virus (AcNPV, U.S. Pat. No. 5,731,182) or recombinant AcNPV derived vectors also engineered to express the envelope protein of vesicular stomatitis virus (here termed xe2x80x9cAcNPV greater than VSVGxe2x80x9d) (Barsoum et al.,1997, Hum Gene Ther 8:2011-2018); and (c) transposon based vectors for example Himar1, piggyBac, Hermes, hobo, minos, mariner, etc.
The constructs may also include a variety of other components as dictated by practical or experimental objectives. For example, in particular embodiments, the constructs contain one or more of the following elements:
(1) a xe2x80x9ctest genexe2x80x9d operably fused to a promoter whose function is to be assayed in the transgenic animal, e.g. as a possible biopesticide or pesticide target;
(2) a xe2x80x9cproduct genexe2x80x9d operably fused to a promoter, which produces a useful product which can be isolated from transgenic animals, e.g. a modified silk gene or biopharmaceutical;
(3) a xe2x80x9ctransformation genexe2x80x9d operably fused to a promoter, which alters the physical or behavioral properties of transgenic animals in useful ways, e.g. a xe2x80x9cpacification genexe2x80x9d which tames africanized bees or fire ants;
(4) a xe2x80x9cpromoter-less or enhancer-less reporter genexe2x80x9d for gene tagging and mutagenesis;
(5) a regulatable xe2x80x9cenhancerxe2x80x9d or xe2x80x9cpromoterxe2x80x9d to drive expression of genes adjacent to the insertion site of the vector for misexpression analysis;
(6) a xe2x80x9cDNA manipulation elementxe2x80x9d such as a recombinase action site (e.g. FRT or loxP sites) for engineering specific chromosomal rearrangements, insertions, or deletions in transgenic animals; and
(7) an xe2x80x9cinsulator elementxe2x80x9d which protects transgenes from interfering regulatory effects or position effects of adjacent enhancers or silencers.
The contructs are introduced the target animal or cell genome by any convenitent method, as advised by the nature of the construct and target animal or cell; well-established methodologies include microinjection, electroporation, lipofection, biolistics and the like, and genome site integration may be targeted (e.g. by homologous recombination) or nontargeted. Following introduction of the construct into the target animal genome, the methods involve selecting for transgenesis by visually detecting the marker in a photoreceptive cell or organ of the animal. A variety of means may be used to detect the marker, depending on the marker, animal, requisite throughput, etc. Detection may be indirect, by detecting a colored or fluorescent catalytic product of the maker, or preferably, direct, by detecting a colored or fluorescent marker. As used herein, xe2x80x9cvisual detectionxe2x80x9d, and variants thereof, means detecting changes in the emission or reflection of light by direct and/or indirect means including visual inspection, visual inspection enhanced by the use of an optical instrument such as a microscope, photographic or photochemical measurement, photoelectric measurement, etc. For example, detection may be facilitated by automated and/or robotic instrumentation such as fluorimeters, digital imaging spectroscopy (Delagrave et al., (1995) Biotechnology 13:151-154; Youvan et al., (1995) Methods Enzymol 246:732-748; U.S. Pat. No. 5,852,498), etc. Table 7 shows successful detection of markers of transgenesis pursuant to the subject methods.